Total Protein Phosphatase 2A Activity Research Articles

Microcystin-LR promotes cell proliferation in the mice liver by activating Akt and p38/ERK/JNK cascades

Microcystin-LR (MC-LR), a heptapeptide produced by blue-green algae, is shown to induce cytotoxicity by inhibiting protein phosphatase 2A (PP2A) activity. Our previous study revealed that MC-LR promoted cell proliferation in vitro by activating the Akt/mTORC1/S6K1 pathway. This study aims to further investigate the effects of MC-LR on cell proliferation and the correlated mechanisms in vivo. Mice were injected intraperitoneally with 20–80 μg/kg/d MC-LR from 2 h (hours) to 4 d (days). The results showed that the associations of MC-LR with PP2A/C (PP2A C subunit) were concentration-dependent but not time-dependent in the liver, whereas the total PP2A activity was inhibited in both concentration and time dependent manners. The PP2A regulator α4 was found to release its associated PP2A/C as MC-LR bound to PP2A/C. Importantly, 80 μg/kg MC-LR promoted liver cell proliferation beginning at 1 d post exposure, and hyperproliferation also occurred in the 40 μg/kg group at 4 d after exposure. Meanwhile, the Akt/mTORC1/S6K1 and Akt/β-catenin signaling pathways were activated as early as at 2 h post exposure. Furthermore, MC-LR also activated ERK/p38/JNK MAPKs as early as at 2 h post exposure, which was supported by the hyperphosphorylation of their substrates, ATF-2, c-Jun and c-Myc. Interestingly, the total c-Jun and c-Myc levels also increased after MC-LR exposure. These findings indicate that MC-LR can also promote cell proliferation in vivo, and the activation of Akt and MAPK signaling pathways due to PP2A inhibition is proposed to participate in this process.

The PP2A Subunit B55α Suppresses Survival Signaling in Acute Myeloid Leukemia Cells

Abstract 396Acute myeloid leukemia (AML) is still highly fatal so a better understanding of the mechanisms controlling chemoresistance is critical for the development of more effective therapies. The Protein Kinase B (AKT) regulates diverse cellular functions including protein translation, transcription, cell proliferation and apoptosis and is emerging as an important target for AML therapy. We have reported that activation of AKT predicts poor clinical outcome for patients with AML. Mutations of upstream activators of AKT such as Phosphatidylinositol 3 Kinase (PI3K) and RAS are well characterized and have been implicated in many cancers including AML. However, signal transduction is a dynamic process and the possibility arises that loss of protein phosphatase function can promote kinase activation in the absence of constitutive activation of the kinase by upstream activators. AKT has been shown to be regulated by Protein Phosphatase 2A (PP2A). PP2A is a family of hetero-trimeric serine/threonine phosphatases. While family members share a fairly common catalytic core (i.e. A and C subunits), the functional component of each PP2A isoform lies in its regulatory subunit (i.e. B subunit). Recent data suggests that suppression of PP2A can promote activation of survival signaling including the activation of AKT in AML. We recently identified that low expression of the PP2A isoform that acts as the AKT phosphatase (i.e. the one containing the B55α subunit) in AML negatively correlates with phosphorylation of AKT at threonine 308 and with AKT activation in a cohort of 511 AML patients (Ruvolo et al Leukemia, 2011). Patients with low B55α expression had shorter complete remission duration. In the current study, we now found by Reverse Phase Protein Analysis (RPPA) that there is a negative correlation between B55α expression and expression and phosphorylation of a number of survival kinases including Protein Kinase C a (PKCα) and SRC in AML patients. PP2A has been shown to negatively regulate the PKC family of kinases but this is the first report to implicate B55α in that process. While levels of phosphorylated PKCα negatively correlated with B55α, total levels of the kinase also were negatively correlated with the B subunit suggesting that phosphatase likely does not dephosphorylates PKCα but rather suppresses expression of the kinase. While SRC has not been linked to B55α, the kinase has been shown to negatively regulate the PP2A catalytic subunit (PP2A/C). Since PP2A is an obligate hetero-trimer, inactivation of PP2A/C via SRC could result in proteolytic cleavage of the B subunit. Consistent with this notion, treatment of OCI-AML3 cells with the tyrosine kinase inhibitor Dasatinib resulted in reduction of PP2A/C levels. Consistent with a mechanism whereby SRC regulated B55α rather than vice-versa, OCI-AML3 cells expressing B55α shRNA did not display changes in SRC phosphorylation status though there was a >67% loss of the B subunit in these cells. To determine the role of the AKT Phosphatase in AML cell survival, the OCI-AML3 cells expressing B55α shRNA were used to determine if reduction of levels of the phosphatase would promote AKT activation and promote chemoresistance. Cells with B55α shRNA exhibited higher levels of AKT S473 phosphorylation suggesting activation of the kinase. Furthermore, OCI-AML3 cells expressing B55α shRNA were more resistant to killing by the conventional chemotherapy agent AraC. One surprising finding was that OCI-AML3 B55α shRNA transductant cells were more resistant to okadaic acid, a PP2A inhibitor that is highly toxic to AML cells. Despite effects on AKT activation and survival, the reduction of the B subunit had no effect on total PP2A activity as determined by molybdate dye assay. Reduction of B55α did promote expression of at least one PP2A B regulatory subunit (i.e. B56α levels increased nearly 2 fold). Interestingly, B56α is positively regulated by AKT suggesting a possible feedback mechanism may exist between AKT and the various PP2A isoforms. An intriguing possibility arises that while PP2A is generally considered a tumor suppressor, inhibition of the AKT Phosphatase may activate an okadaic acid resistant PP2A isoform with survival function. Taken together, these results suggest that B55α may suppress survival signaling by kinases such as AKT and loss of function of the B subunit may promote resistance to chemotherapy in AML. Disclosures:No relevant conflicts of interest to declare.

Molecular functions of the PP2A regulatory subunit Tap46 in plants

Tap42/α4 is a regulatory subunit of the protein phosphatase 2A (PP2A) family of phosphatases and plays a role in the target of rapamycin (TOR) pathway that regulates cell growth, ribosome biogenesis, translation and cell cycle progression in both yeast and mammals. We determined the cellular functions of Tap46, the plant homolog of Tap42/α4, in both Arabidopsis thaliana and Nicotiana benthamiana. Tap46 associated with the catalytic subunits of PP2A and the PP2A-like phosphatases PP4 and PP6 in vivo. Tap46 was phosphorylated by TOR in vitro, indicating that Tap46 is a direct substrate of TOR kinase. Tap46 deficiency caused cellular phenotypes that are similar to TOR-depletion phenotypes, including repression of global translation and activation of both autophagy and nitrogen recycling. Furthermore, Tap46 depletion regulated total PP2A activity in a time-dependent manner similar to TOR deficiency. These results suggest that Tap46 acts as a positive effector of the TOR signaling pathway in controlling diverse metabolic processes in plants. However, Tap46 silencing caused acute cell death, while TOR silencing only hastened senescence. Furthermore, mitotic cells with reduced Tap46 levels exhibited chromatin bridges at anaphase, while TOR depletion did not cause a similar defect. These findings suggest that Tap46 may have TOR-independent functions as well as functions related to TOR signaling in plants.

Adenosine A1 and A2A receptor regulation of protein phosphatase 2A in the murine heart

Adenosine plays a role in regulating the contractile function of the heart. This includes a positive ionotropic action via the adenosine A(2A) receptor (A(2A)R) and an inhibition of beta(1)-adrenergic receptor-induced ionotropy (antiadrenergic action) via the adenosine A(1) receptor (A(1)R). Phosphatase activity has also been shown to influence contractile function by affecting the level of protein phosphorylation. Protein phosphatase 2A (PP2A) plays a significant role in mediating the A(1)R antiadrenergic effect. The purpose of this study was to investigate the effects of A(2A)R and A(1)R on the activities of PP2A in hearts obtained from wild-type (WT) and A(2A)R knockout (A(2A)R-KO) mice. PP2A activities were examined in myocardial particulate and cytoplasmic extract fractions. Treatment of wild-type hearts with the A(1)R agonist CCPA increased the total PP2A activity and increased the particulate:cytoplasmic PP2A activity ratio. Treatment with the A(2A)R agonist CGS-21680 (CGS) decreased the total PP2A activity and decreased the particulate:cytoplasmic PP2A activity ratio. This indicated a movement of PP2A activity between cell fractions. The effect of CCPA was inhibited by CGS. In A(2A)R-KO hearts the response to A(1)R activation was markedly enhanced whereas the response to A(2A)R activation was absent. These data show that A(2A)R and A(1)R regulate PP2A activity, thus suggesting an important mechanism for modulating myocardial contractility.

A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression

Activity of protein phosphatase 2A (PP2A) is tightly regulated and performs a diverse repertoire of cellular functions. Previously we isolated a dominant-negative active site mutant of the PP2A catalytic (C) subunit using a yeast complementation assay. We have established stable fibroblastic cell lines expressing epitope-tagged versions of the wild-type and H118N mutant C subunits and have used these cells to investigate mechanisms that regulate PP2A activity. Cells expressing the mutant C subunit exhibit a decreased growth rate and a prolonged G1 cell cycle phase. The mutant protein is enzymatically inactive, but extracts made from cells expressing the H118N C subunit show normal levels of total PP2A activity in vitro. The H118N mutant shows reduced binding to the regulatory A subunit, but binds normally to the alpha4 protein, a non-canonical regulator of PP2A. Expression of the H118N mutant interferes with the normal control of C subunit abundance, causing accumulation of the endogenous wild-type protein as well as the mutant transgene product. Our results indicate that the H118N mutant isoform retards C subunit turnover and suggest that PP2A C subunit turnover may be important for normal cell cycle progression.

Critical Role for Protein Phosphatase 2A Heterotrimers in Mammalian Cell Survival

The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (alpha/beta), whereas three gene families (B, B', and B'') with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aalpha subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aalpha RNAi decreased total PP2A activity as well as protein levels of C, B, and B' but not B'' subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aalpha triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aalpha cDNAs rescued RNAi-induced loss of the C subunit, and Aalpha point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Abeta and Aalpha mutants capable of binding to at least one family of regulatory subunits were able to delay Aalpha RNAi-induced death of PC12 cells. However, only the expression of wild-type Aalpha restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aalpha subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.

Glucocorticoid Regulation of Calcium-activated Potassium Channels Mediated by Serine/Threonine Protein Phosphatase

Adrenal glucocorticoids exert powerful effects on cellular excitability in neuroendocrine cells and neurons, although the underlying mechanisms are poorly understood. In metabolically intact mouse anterior pituitary corticotrope (AtT20) cells glucocorticoid-induced proteins render large conductance calcium-activated potassium (BK) channels insensitive to inhibition by protein kinase A (PKA). In this study we have addressed whether this action of glucocorticoids is mediated via protein phosphatase activity at the level of single BK channels. In isolated inside-out patches from control AtT20 cells BK channels (125 pS) were inhibited by activation of closely associated PKA. Pretreatment (2 h) of cells with 1 microM dexamethasone before patch excision did not modify the intrinsic properties or expression levels of BK channel alpha-subunits in AtT20 cells. However, PKA-mediated inhibition of BK channel activity in isolated patches from steroid-treated cells was severely blunted. This effect of steroid was not observed using adenosine 5'-O-(3-thiotriphosphate) as phosphate donor or on exposure of the intracellular face of the patch with 10 nM of the protein phosphatase inhibitors okadaic acid or calyculin A but was mimicked by application of protein phosphatase 2A (PP2A) to the intracellular face of patches from control cells. Glucocorticoids did not modify total PP2A activity in AtT20 cells, suggesting that modified PP2A-like phosphatase activity closely associated with BK channels is required for glucocorticoid action.

Increase in liver protein phosphatase-1 in spontaneously diabetic Chinese hamsters

Two broad-specifically protein phosphatases, termed protein phosphatase-1 (PrP-1) and protein phosphatase-2A (PrP-2A), accounting for all the hepatic activity regulating glycogen phosphorylase, were measured in spontaneously diabetic Chinese hamsters exhibiting persistent glycosuria. When compared with genetically related inbred sublines free of glycosuria, diabetic animals demonstrated approximately 25% increase in PrP-1 activity measured either in crude tissue extracts or in cytosols fractionated by ion-exchange chromatography. No significant alteration in total PrP-2A activity was observed in the diabetic animals. These findings indicate that a specific change in hepatic PrP-1 is associated with genetically acquired diabetes in Chinese hamsters. In contrast to reported data using animals with experimentally induced diabetes mellitus, hepatic PrP-1 was increased in the spontaneously diabetic Chinese hamsters. The data suggests that distinct alterations in PrP-1 and associated metabolic consequences are exhibited by different types of diabetes.